【摘要】 目的:观察氢醌对HL-60细胞向单核细胞分化的影响,并初步探讨miR-146a的调控作用。方法:设miR-146a-5p抑制剂和阴性对照处理的HL-60细胞,分别以不同浓度(0、0.5、1.0、2.5和5.0μmol/L)的氢醌处理3 h后,接种于含(PMA)的培养基中,培养72 h诱导其分化。采用实时荧光定量PCR检测细胞内miR-146a及其靶基因的表达量,流式细胞术检测CD11b和CD14的表达,Western blot检测TRAF6蛋白及其下游调控因子IκBα蛋白的表达水平。结果:与对照组相比(0μmol/L),氢醌可抑制PMA诱导的HL-60细胞CD11b和CD14的表达,5.0μmol/L的氢醌使其表达量分别减少44%和38%(P均<0.01);同时miR-146a的表达比对照组升高近2倍(P<0.01);TRAF6 mRNA的表达下降约40%,其蛋白表达下降74%(P均<0.01);磷酸化IκBα蛋白的表达减少约45%,IκBα总蛋白表达升高约73%(P均<0.01)。当抑制miR-146a的表达后,氢醌对TRAF6、IκBα和磷酸化IκBα蛋白表达的影响不明显。结论:miRNA-146a是氢醌影响HL-60细胞分化的调控因子之一。更多还原

【Abstract】 OBJECTIVE: To study the effects of hydroquinone(HQ) on monocytic differentiation of HL-60 cells and explore the regulatory role of miR-146a in the mechanism of toxicity. METHODS: Using the model of monocytic differentiation induction with phorbol 12-myristate 13-acetate(PMA),HL-60 cells were transfected with miR-146a-5p inhibitor or negative control and then treated with various doses of hydroquinone(0,0.5,1.0,2.5 and 5.0 μmol/L) for 3 h,and cells collected after induction with PMA for 72 h. The expressions of miR-146a and its target gene TRAF6,were measured by real-time quantitative PCR. CD11b and CD14 levels were also determined by flow cytometry. Western blot was used to detect protein expression levels of TRAF6,and its downstream regulatory factor IκBα. RESULTS: Hydroquinone could inhibit the expressions of CD11b and CD14 in HL-60 cells induced by PMA. Compared with control group(0 μmol/L),5.0 μmol/L of hydroquinone decreased the expressions of CD11b and CD14 by 44% and 38%,respectively(both P <0.01);the expression of miR-146a increased nearly 2-fold(P<0.01). The expressions of TRAF6 mRNA and protein were decreased by 40% and 74%(P<0.01),respectively. The expression of phosphorylated IκBα protein was reduced by approximately 45%,and the total IκBα protein increased nearly 73%(P<0.01). After inhibiting the expression of miR-146a,there were no significant effects of hydroquinone on TRAF6,phosphorylated IκBα and total IκBα protein expression. CONCLUSION: miRNA-146a may be one of the regulatory factors in whichhydroquinone influence HL-60 cells differentiation.更多还原